Broad, long-term objectives: To create powerful, sensitive techniques for understanding the many roles of peptides in the brain. To apply these tools to peptides that are important in protecting, damaging or regenerating neurons following conditions such ischemia or seizures. To make quantitative measurements of the activity of neuropeptide-inactivating peptidases in living tissue. Specific Aims: 1. Develop electroosmotic approaches for sampling extracellular fluid from living tissue (e.g., organotypic nippocampal slice cultures). It can also be operated to both introduce and sample (electroosmotic push-pull). It will be used to measure peptide concentrations and peptidase activity in living tissue. 2. Determine glutathione (extracellular) and the rate of its breakdown by gamma-glutamyl transpeptidase in organotypic hippocampal cultures. Electroosmotic sampling will be interfaced to online analysis for the antioxidant peptide, glutathione. 3. Determine the rate of breakdown of the neuroprotective peptide galanin. This includes improving detection limits for peptides through better separation and detection. Using the spatial resolution inherent in the new sampling approach, determine the rate of galanin breakdown in various areas of the hippocampus. Test the hypothesis that procedures known to protect neurons in stroke-like conditions decrease the activity of enzymes that inactivate galanin. Sample splitting postcolumn with nanofraction collection allows for offline mass spectrometric confirmation of peak identity. 3. Determine the rate of degradation of the neurotrophic factor BDNF in living tissue. Health relatedness: About 700,000 people will have a stroke annually in the US. One quarter will die. One fifth will suffer lasting disability. It is now known that the nerve cell damage that occurs following stroke is due to a complex biochemical process taking hours to days. Several peptides, like galanin and BDNF are known to be neuroprotective. Increasing their lifetime in the brain and their extent of influence post trauma can improve survival and outcome